General Discussion of the Background
Halogenated aromatic compounds have been produced industrially on a large scale for several decades. Brominated aromatic compounds have found use as flame retardants, and fluorinated and iodinated aromatic compounds are components of pharmaceutical agents. The chlorinated aromatics have been widely used as pesticides (for example, 2,4-dichlorophenoxyacetic acid and pentachlorophenol) and in industrial settings (for example, polychlorinated biphenyls are employed in electrical equipment and as hydraulic fluids). Typically, these compounds are highly chemically inert, hydrophobic and toxic. Their widespread distribution in the environment is therefore of great concern.
Halogenated phenols comprise a significant subgroup of the halogenated aromatics. The halogenated phenols include pentachlorophenol and its salt, sodium pentachlorophenol. These two compounds (hereinafter referred to collectively as PCP) are among the most widely distributed biocides used in the United States. PCP is an inhibitor of oxidative phosphorylation and, as such, is lethal to a wide variety of organisms, including both plants and animals. PCP is primarily employed by the wood preserving industry as a fungicide and pesticide and is also used in products such as herbicides and disinfectants.
PCP is highly toxic and tends to persist in the environment and within food chains. The adverse effects of low-level PCP contamination on organisms such as fish, shrimp, oysters, clams, and rats are well documented and are reviewed in Rao (1977). A number of studies conclude that contamination of tissues of human populations with PCP at a level of 10 to 20 parts per billion is average for industrialized societies (Rao, 1977). PCP is thought to be mutagenic or at least co-mutagenic, and human exposure to PCP poses significant health hazards.
Removal of PCP and other halogenated aromatics from contaminated environments is therefore an issue of great concern. Methods such as the use of activated charcoal to decontaminate water are currently employed, and efforts have been directed towards the development of bioremediation approaches. Several reports have documented the ability of mixed microbial populations to degrade PCP and other halogenated aromatics. These microbes may be used in combination with existing bioremediation processes and techniques to remove PCP and other halogenated aromatics from contaminated water supplies and soils. Suitable bioremediation techniques include those disclosed in several patents, including U.S. Pat. No. 4,713,340 to Crawford, and East German patent No. 3,601,979 to Debus and Rohde, which describe methods for inoculating bacteria into contaminated environments or materials, including soils and water, and providing conditions under which the inoculated bacterial populations break down the contaminating halogenated aromatics. Another mode of use for such bacteria is disclosed in Japanese patent No. 49094569 to Nishimura in which a crude enzyme extract from a gram-negative bacillus is used to degrade polychlorinated biphenyl.
Further investigations have led to the isolation and characterization of pure strains of bacteria able to degrade halogenated aromatics. In particular, several bacterial strains have been isolated that are capable of breaking down PCP into cellular metabolites. These strains include Arthrobacter sp. Strain ATCC 33790 (Schenk et al., 1989), Rhodococcus chlorophenolicus PCP-I (DFN 43826) (Apajalahti and Salkinoja-Salonen, 1987), and Flavobacterium sp. Strain ATCC 39723 as described in U.S. Pat. No. 4,713,340 to Crawford.
The mechanisms by which such microorganisms degrade PCP and other halogenated aromatics are not completely understood. Proposed mechanisms of biodegradation of halogenated aromatics are discussed in Commandeur and Parsons (1990). Microbial metabolism of PCP is addressed in Reiner et al. (1978), and the transformation of PCP under environmental conditions including microbial breakdown has been reviewed by Engelhardt et al. (1986). Based on the isolation of metabolites extracted from culture media, various degradation pathways have been proposed. These pathways have in common the step by step dechlorination of PCP to less chlorinated compounds after which the aromatic ring is cleaved.
Studies of cell extracts from Rhodococcus chlorophenolicus have proposed that the initial step of PCP breakdown is hydroxylation to yield tetrachlorohydroquinone (Apajalahti and Salkinoja-Salonen, 1987a) which is subsequently converted to dichlorotrihydroxybenzene by a reaction involving both hydrolytic and reductive dechlorinations (Apajalahti and Salkinoja-Salonen, 1987b). Further dechlorinations then yield 1,2,4 trihydroxybenzene. Trihydrochloroquinone was found to be degraded only very slowly, suggesting that it is not an intermediate in the pathway.
A pathway involving initial hydrolytic dechlorination in the para position of PCP to form 2,3,5,6-tetrachloro-p-hydroquinone (TeCH) and further reductive dechlorinations has been proposed to be responsible for the degradation of PCP by the aerobic Flavobacterium sp. Strain ATCC 39723 (Steiert and Crawford, 1986). This strain is also able to degrade and dechlorinate a range of di, tri and tetrachlorophenols (Steiert et al., 1987). Chlorophenols with chlorine substituents in both ortho (2 and 6) positions were degraded most readily. Of these, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol and PCP were inducers of the complete PCP degradation pathway.
The microbial breakdown of PCP is thus generally considered to proceed by hydroxylation and dechlorination. The microbes exhibiting PCP breakdown activity must produce enzymes which are able to cleave the carbon-chloride bonds in PCP. However, these and other proteins involved in PCP breakdown have been neither characterized nor purified. Similarly, the genes encoding these enzymes have yet to be identified and cloned.
It is an object of this invention to characterize the process of bacterial PCP breakdown and by so doing to provide information and materials of utility for novel bioremediation technologies.
Specifically, it is an object of this invention to identify and purify enzymes involved in the bacterial breakdown of PCP.
It is another specific object of this invention to identify and clone the genes encoding such enzymes.
It is another object of this invention to provide recombinant DNA vectors which contain the enzymes involved in the bacterial breakdown of PCP, which vectors are capable of directing the expression of these enzymes in heterologous bacterial hosts.
It is a further object of this invention to provide bacteria which are stably transformed with these vectors and which express one or more of the enzymes involved in the bacterial breakdown of PCP.
These and other objects of the invention will be understood more clearly by reference to the following detailed description.